Publications by authors named "Mariangela Del Vecchio"

6 Publications

  • Page 1 of 1

SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains.

PLoS Pathog 2014 May 8;10(5):e1004124. Epub 2014 May 8.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
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http://dx.doi.org/10.1371/journal.ppat.1004124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014459PMC
May 2014

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.

FASEB J 2013 Dec 20;27(12):4723-30. Epub 2013 Aug 20.

1Centro Ricerche Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

NarE is an arginine-specific mono-ADP-ribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NAD-concentration-dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Site-directed mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R(7) residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.
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http://dx.doi.org/10.1096/fj.13-229955DOI Listing
December 2013

EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation.

mBio 2013 Jul 23;4(4). Epub 2013 Jul 23.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Unlabelled: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site.

Importance: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.
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http://dx.doi.org/10.1128/mBio.00206-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3735183PMC
July 2013

Arginine-specific mono ADP-ribosylation in vitro of antimicrobial peptides by ADP-ribosylating toxins.

PLoS One 2012 7;7(8):e41417. Epub 2012 Aug 7.

Novartis Vaccines & Diagnostics, Siena, Italy.

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041417PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413682PMC
January 2013

Identification of an iron-sulfur cluster that modulates the enzymatic activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase.

J Biol Chem 2009 Nov 10;284(48):33040-7. Epub 2009 Sep 10.

Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.

In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys(67) and Cys(128) into serine caused a drastic reduction in the E(420)/E(280) ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes.
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http://dx.doi.org/10.1074/jbc.M109.057547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785144PMC
November 2009

Mono ADP-ribosylation inhibitors prevent inflammatory cytokine release in alveolar epithelial cells.

Mol Cell Biochem 2008 Mar 9;310(1-2):77-83. Epub 2007 Dec 9.

Novartis Vaccines and Diagnostics, Via Fiorentina, 1, 53100 Siena, Italy.

A549, a type II alveolar epithelial cell line stimulated with LPS (10 mug/ml), released high levels of the inflammatory cytokines IL-6 and IL-8. Here, we have investigated whether ADP-ribosylation inhibitors block the LPS-triggered cytokine release in epithelial cells. When coincubating A549 with LPS and meta-iodobenzylguanidine or novobiocin, selective arginine-dependent ART-inhibitors, the release of IL-6 and IL-8 was inhibited in a concentration-dependent manner. This effect has been linked with the presence of a functionally active arginine ADP-ribosylating enzyme on the cell surface. To this aim, we amplified by RT-PCR the ART1 transcript and identified four ADP-ribosylated proteins likely substrate for ART1. The mechanism behind the cytokine inhibition in epithelial cells seems to be correlated with the presence of ART1, which behaves as an essential positive regulator of inflammatory cytokines. This novel observation indicates this enzyme as well as other novobiocin/MIBG sensitive ARTs as potential targets for the development of new therapeutic strategies.
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http://dx.doi.org/10.1007/s11010-007-9667-3DOI Listing
March 2008